BRCC3 mediates inflammation and pyroptosis in cerebral ischemia/reperfusion injury by activating the NLRP6 inflammasome

Abstract Aims Neuroinflammation and pyroptosis are key mediators of cerebral ischemia/reperfusion (I/R) injury‐induced pathogenic cascades. BRCC3, the human homolog of BRCC36, is implicated in neurological disorders and plays a crucial role in neuroinflammation and pyroptosis. However, its effects and potential mechanisms in cerebral I/R injury in mice are unclear. Methods Cellular localization of BRCC3 and the interaction between BRCC3 and NLRP6 were assessed. Middle cerebral artery occlusion/reperfusion (MCAO) and oxygen–glucose deprivation/reoxygenation (OGD/R) models were established in mice and HT22 cells, respectively, to simulate cerebral I/R injury in vivo and in vitro. Results BRCC3 protein expression peaked 24 h after MCAO and OGD/R. BRCC3 knockdown reduced the inflammation and pyroptosis caused by cerebral I/R injury and ameliorated neurological deficits in mice after MCAO. The effects of BRCC3 on inflammation and pyroptosis may be mediated by NLRP6 inflammasome activation. Moreover, both BRCC3 and its N‐ and C‐terminals interacted with NLRP6, and both BRCC3 and its terminals reduced NLRP6 ubiquitination. Additionally, BRCC3 affected the interaction between NLRP6 and ASC, which may be related to inflammasome activation. Conclusion BRCC3 shows promise as a novel target to enhance neurological recovery and attenuate the inflammatory responses and pyroptosis caused by NLRP6 activation in cerebral I/R injury.

basis of the inflammatory response in the central nervous system.
Activating the inflammatory response results in the release of large amounts of inflammatory factors, which promote the development of secondary brain injury. 5 a part of innate immunity, the NLR family 6,7 can recognize pathogen-associated molecular patterns and damage-associated molecular patterns in the cytoplasm after tissue damage or cell death and is part of the pattern recognition receptor family involved in inflammasome formation. 8,9Multiple NLR family members regulate inflammatory responses in the central nervous system.
2][13] Upon activation, NLRP6 assembles with adapter ASC and effector pro-caspase-1 to form an inflammasome and further mediates caspase-1 activation. 14,15Activated caspase-1 cleaves the pro-inflammatory cytokines IL-1β and IL-18 into their mature forms through proteolysis and mediates the cleavage and activation of membrane pore-forming protein gasdermin D (GSDMD), leading to the release of cytosolic contents, including the mature forms of IL-1β and IL-18, 16,17 which trigger pyroptosis.However, the specific mechanism underlying NLRP6 activation in cerebral I/R injury requires further investigation.
BRCC3, the human homolog of BRCC36, specifically recognizes and hydrolyzes ubiquitin chains formed via K63 links, reducing substrate ubiquitination modification levels; thus, it is also called ubiquitin hydrolase. 18BRCC3 can activate inflammasomes to mediate the inflammatory response. 19BRCC3 activates the NLRP6 inflammasome in rats. 20However, the effect of BRCC3 in deubiquitinating NLRP6 and its specific mechanism of action in activating NLRP6 during cerebral I/R injury in mice remain poorly understood.Therefore, investigating these aspects will provide deeper insights into the involvement of BRCC3 in neuroinflammation and pyroptosis, potentially paving the way for novel therapeutic strategies to mitigate the harmful effects of cerebral I/R injury and improve the neurological outcomes in patients.
We aimed to explore the effect of BRCC3 in neuroinflammation and pyroptosis after cerebral I/R injury in mice and the potential mechanism via which BRCC3 activates the NLRP6 inflammasome.

| Animals and cell culture
All experimental protocols and procedures were approved by the Ethical Committee of Chongqing Medical University, following the guidelines of the Experimental Animal Institute of Chongqing Medical University.A total of 153 male C57BL/6J mice (Chongqing Medical Animal Experimentation Center) weighing 18-22 g were used.All animals were housed under a 12-h light-dark cycle at 21-26°C with unrestricted access to food and water.

| MCAO model
The MCAO surgical procedure was performed as previously described. 21The mice were intubated and kept under 3% isoflurane anesthesia (v/v in air).An incision was made at the neck midline under supine position.A nylon monofilament (AL1800; Jialing, Guangzhou, China) was inserted into the middle cerebral artery via the left external carotid artery for 1 h.The nylon monofilaments were withdrawn, and reperfusion was performed for 24 h.The Sham group was anesthetized, and the common carotid artery was located and sutured without embolization.

| Laser Doppler flowmeter
The changes in cerebral blood flow were monitored using a laser Doppler flowmeter (LDF) (PeriFlux System 5000; Perimed, China) during MCAO modeling.We fixed the anesthetized mice to the locator, exposed the coronal and sagittal sutures, and used the intersection of the two sutures as the coordinate origin.We selected 1 mm to the left and 0.3 mm to the back for positioning.We detected a stable blood flow using an LDF after fixing the probe in place and before inserting the embolus into the internal carotid artery.The modeling was considered successful if the LDF decreased to 70%.

| Cerebral infarct volume evaluation
To determine the volume of cerebral infarction, the entire brains of the mice were removed and kept at −20°C for 30 min before being sliced at 2 mm intervals (5 slices) as previously reported. 22The frozen sections were submerged in 2% 2,3,5-triphenyltetrazolium chloride (TTC) (37°C; 15 min); this step necessitates protection from light.The samples were then fixed in 4% paraformaldehyde at 4°C for a period of 48 h.Images were obtained utilizing a digital camera and analyzed with ImageJ analysis software.Infarct volume was calculated as follows: {[total lesion volume − (ipsilateral hemisphere volume-contralateral hemisphere volume)]/contralateral hemisphere volume} × 100%.

| Assessment of the neurological score
According to the literature, nerve injuries were scored blindly 24 h after modeling 23 as follows: 0, normal; 1, inability to fully extend the contralateral forepaw; 2, turning to the contralateral side during locomotion; 3, tilted to the contralateral side; 4, inability to locomote spontaneously; 5, dead.Mice with a score of 0 and 5 were excluded.

| Hematoxylin-eosin (HE) and Nissl staining
According to a previous study, 24 after MCAO, the brains were rapidly collected and preserved in 4% paraformaldehyde for roughly 24 h.Next, coronal brain tissue sections, 2 mm thick, were cut from the whole brains and dehydrated.The sections were embedded in paraffin wax, stained with HE and Nissl, and observed under a microscope.Nissl-stained neurons were quantitatively analyzed utilizing ImageJ software.
Oxygen and glucose deprivation/reoxidation (OGD/R) 25 experimental cells were placed in DMEM without glucose and cultured for 4 h in a Tri-Gas incubator containing 1% O 2 , 94% N 2 , and 5% CO 2 .
Glucose-free DMEM was then replaced with a normal high-glucose culture medium (DMEM containing 1% penicillin/streptomycin and 10% FBS), and the cells were cultured in a 5% CO 2 incubator for 24 h.The cells in the control group were cultured normally without treatment.

| Experimental design
Animals and cells were grouped as shown in the Supplementary Material S1.

| Experiment 1
Western blotting was used to determine the time course of endogenous BRCC3 expression in the left cerebral cortex.
Immunofluorescence staining was performed 24 h after MCAO to determine the cellular localization of BRCC3.

| Experiment 2
To assess the effects of BRCC3 on neuroinflammation and pyroptosis, we knocked down BRCC3 using siRNA in mouse brain tissue 24 h before MCAO.We established 24 h cerebral I/R models in mice for detection.The grouping was as follows: Sham, MCAO, MCAO+NC, and MCAO+si-BRCC3.

| Experiment 3
The time course of endogenous BRCC3 in HT22 cells was detected using western blotting analysis.

| Experiment 4
To elucidate the role of BRCC3 in neuroinflammation and pyroptosis, BRCC3 siRNA dissolved in diethyl pyrocarbonate (DEPC) was transfected into HT22 cells 24 h before OGD/R.To detect related markers, we established 24 h oxygen-glucose-deprivation/reoxygenate.

| Experiment 5
To assess whether BRCC3 affects downstream inflammation and pyroptosis through NLRP6, a lentiviral-packaged BRCC3overexpression plasmid was transfected into HT22 cells before OGD/R.To detect related markers, we established 24 h oxygen-glucose-deprivation/reoxygenation.

5'-ACGUGACACGUUCGGAGAATT-3′).
Firstly, we dissolved siRNA in DEPC water.After the mice were completely anesthetized, they were fixed on a stereotaxic device.
After determining the location of the Bregma point, the mice were injected via the injection point.The position of the first injection point A was 0.3 mm behind the Bregma point, 1.0 mm beside the central axis, and 2.5 mm below the skull surface (coordinates: x = 0.3, y = 1.0, and z = 2.5).The coordinates of the second and third injection points were B (x = 0.5, y = 1.3, and z = 2.2), and C (x = 0.3, y = 2.2, and z = 2.2), respectively.The injection volume was 2.5 μL.The injection was performed over 5 min.After 10 min, the needle was slowly removed.
We inoculated the HT22 cells into 6-well plates at a density of 1.5 × 10 5 /mL, 24 h before transfection.Before adding the transfection complex, HT22 cells were provided with a fresh culture medium.
The transfection complex (Opti-MEM; 125 μL, BRCC3-siRNA; 5 μL, and Lipo8000™; 5 μL) was gently mixed, reacted at room temperature for 20 min, and then 125 μL mixture was added to each well.All were thoroughly mixed and placed into an incubator set at 37°C.OGD/R was performed after 24 h.

| Western blot analysis
Equal amounts and volumes of protein samples (around 20-50 μg) were added into sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel wells.Firstly, we used different voltages to perform electrophoresis of the proteins based on the gels' protein concentration and separation ability.Electrophoresis was stopped when the bromophenol blue dye had migrated three-quarters of the way through the SDS-PAGE gel.Subsequently, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA) and incubated for 1 h at room temperature with 5% bovine serum albumin.
Following washing with TBST, the membrane was incubated with the corresponding primary antibody in an incubation box (4°C).All primary antibodies used are listed below: anti-BRCC3

| Immunofluorescence staining
The mice were deeply anesthetized 24 h after MCAO and transcardially perfused with 0.9% saline and 4% paraformaldehyde.Whole brains were removed and fixed in 4% paraformaldehyde for 24 h.The brain sections were incubated with primary antibodies overnight at 4°C.

| Plasmid construction and transfection
Primer designs are shown in Supplementary Material S2A.

| Construction of overexpressed plasmid and screening of stable transmissible cell lines
Primer designs are shown in Supplementary Material S2B.
The plasmid construction steps were the same as those described above.Following the successful construction, lentiviral packaging was performed.After packaging was completed, the HT22 cells were infected.After cell passaging, the density reached 70%-80%, and drug screening continued.After the drug screening, the surviving cells were cultured in a purine medium.

| Lentivirus packaging
The lentiviral packaging was performed when the HEK293FT cells reached 70-80% confluency.The lentiviral packaging complex was prepared as follows: Opti-MEM, psPAX2, pMD2.G, and the plasmids were successively added, mixed, and incubated at room temperature for 5 min.PEI was then added, mixed, and incubated at room temperature for 20 min.The HEK293FT cells were digested and passaged at a 1:1 ratio.The above mixture was added to the cells, and the supernatant was collected after 48 h and filtered.

| Co-immunoprecipitation
Cell or tissue proteins were lysed using immunoprecipitation (IP) lysis buffer (containing 10% phosphatase inhibitor and 10% phenylmethyl sulfonyl fluoride).After mixing, the mixture was placed on ice for 30 min.The centrifugal supernatant was collected (4°C, 12000 g, 15 min) and placed on ice.Protein A/G magnetic beads (Beaver, China) were washed with IP binding buffer, then the corresponding IP antibody (50 μg/mL) was added and incubated for 30 min on a mixing rotator.Then, the previously prepared antigen samples were added, and the mixture was mixed with a pipette.After 1 h of reaction on the mixing rotator, magnetic separation was performed, and the mixture was washed with washing buffer.The immune complexes were boiled for 5 min with SDS sample buffer at 95°C and detected by western blotting.

| Statistical analyses
The data were analyzed using GraphPad Prism 8.0.The Shapiro-Wilk test was performed on all data to confirm the normal distribution.Data that met normal distribution were analyzed using one-way analysis of variance (ANOVA) and Tukey's test.Statistical significance was defined as p < 0.05.The mean ± SD was used to express values.Data that did not conform to normal distribution were analyzed using the rank sum test.p < 0.05 indicates a statistically significant difference in the mean rank between the two groups.

| Time course and spatial expression of BRCC3 after MCAO and OGD/R
To determine whether BRCC3 is involved in brain I/R injury, we first monitored cerebral blood flow changes during the model process to ensure successful embolization (Figure 1A).Western blotting was then used to detect endogenous BRCC3 expression in the ipsilateral (left) cerebral cortex.As shown in Figure 1B,C, compared to the Sham group, the BRCC3 levels significantly increased at 6 h, peaked at 24 h after MCAO, and decreased at 48 h.As inflammation peaked after 24 h of reperfusion in the mouse brain ischemic disease process, we chose 24 h as the reperfusion time for subsequent studies.Immunofluorescence staining showed that BRCC3 was more abundant in the neurons than in microglia and astrocytes 24 h after MCAO (Figure 1D).As shown in Figure 1E,F, compared with those in the control group, the BRCC3 levels were significantly increased at 24 h after OGD/R and decreased at 48 h.Therefore, we also chose a 24 h reoxygenation time as the subsequent modeling time for the OGD/R model.

| Effect of BRCC3 on inflammation and pyroptosis after the cerebral I/R Injury
To determine if BRCC3 affects inflammation and pyroptosis dur-

| Influence of BRCC3 on downstream inflammation and pyroptosis through NLRP6 inflammasome
To assess whether BRCC3 regulates downstream inflammation and pyroptosis through the NLRP6 inflammasome, we obtained sta-

| BRCC3 interacts with NLRP6
BRCC3 reportedly monitors downstream inflammation and pyroptosis through the NLRP6 inflammasome.We investigated the relationship between BRCC3 and NLRP6 expression.We transfected BRCC3 and NLRP6 plasmids into HEK293T cells and examined their interactions.The results showed an interaction between BRCC3 and NLRP6, and the N-terminal and C-terminal of BRCC3 were involved (Figure 5A,B).As BRCC3 regulates ubiquitination, we tested whether BRCC3 regulated the ubiquitination level of NLRP6.We found that BRCC3 decreased the ubiquitination of NLRP6 in HEK293T cells and that this effect was associated with both the N-and C-terminal of BRCC3 (Figure 5C,D).Subsequently, we detected the interaction between BRCC3 and NLRP6 in vitro and in vivo (Figure 5E-H).The results showed that BRCC3 and NLRP6 colocalized.We further examined whether they interacted with CO-IP; BRCC3 interacted with NLRP6, and the interaction increased significantly in the I/R model, both in vitro and in vivo.

| Effect of BRCC3 on the activation of NLRP6 inflammasome
Finally, as the assembly of NLRP6 inflammatory bodies reflects NLRP6 inflammasome activation, we further explored the effect of BRCC3 in activating the NLRP6 inflammasome.The presence of BRCC3 significantly improved the interaction between NLRP6 and ASC in HEK293T cells by comparing its N-terminal and C-terminal (Figure 6A,B).Compared with the Sham group, MCAO notably enhanced the binding of NLRP6 and ASC, whereas the BRCC3 siRNA significantly decreased the interaction between NLRP6 and ASC (Figure 6C).

| DISCUSS ION
I/R induces early-stage brain damage associated with oxidative stress, calcium overload, oxygen-free radical damage, and inflammatory cascade response. 26,27Here, the BRCC3 knockdown improved neurological deficits and inflammation, which could lead to reduced brain damage.
This study is the first attempt to investigate the effect of BRCC3 on nerve injury and its potential mechanisms in cerebral I/R in mice.
Our results revealed that: (1) BRCC3 expression was upregulated Cerebral I/R injury induces brain edema, neurological deficits, and neuroinflammation, the primary pathological changes and factors contributing to poor prognosis after MCAO.BRCC3 is extensively expressed in the brain, heart, muscles, kidneys, and small intestine. 28BRCC3 is involved in several brain diseases and plays a significant role. 19,20,29Here, we observed an increase in endogenous BRCC3 expression, soon after MCAO and OGD/R, and BRCC3 was mainly expressed in neurons.This result was consistent with previous findings in a mouse model of Parkinson's disease. 19e NLRP6 inflammasome regulates inflammation during cerebral I/R injury and intracerebral hemorrhage (ICH). 30IL-1β, IL-18, and GSDMD all act downstream of the NLRP6 inflammasome. 31,32CC3 reportedly influences NLRP6 inflammasome activation through interaction with NLRP6 in rats. 20The results of this study The results show that both N-terminal and C-terminal can play a deubiquitination role.The MPN domain located at the N-terminal is a catalytic domain with deubiquitinase activity, 35 which can directly affect the ubiquitination level of NLRP6.However, there is currently no research indicating the specific function and role of C-terminal, which may affect the ubiquitination of NLRP6 through direct or indirect catalysis. 36Later, we will further explore the reasons for the deubiquitination function of both the N-terminal and C-terminal.
Our study had some limitations.BRCC3 is implicated in several pathological processes, such as DNA damage repair, cell apoptosis, angiogenesis, tumorigenesis, bone marrow hyperplasia, myocardial injury, and inflammation. 29,34Here, we examined the effects of brain damage caused by BRCC3 on neuroinflammation and pyroptosis after MCAO in mice.Therefore, we cannot rule out the possibility that BRCC3 mediates other damaging effects in cerebral I/R.
Whether BRCC3 affects NLRP6 through deubiquitination remains unclear and requires further study.
Our findings indicate that BRCC3 may be a novel target for ameliorating cerebral I/R injury and alleviating the inflammatory
injury, we monitored the expression of NLRP6, downstream inflammatory factors, and key components of pyroptosis after BRCC3 knockdown.Compared with that in the Sham group (Figure 3A-F), the MCAO group had increased expression of NLRP6, downstream inflammatory cytokines, and pyroptosis, and BRCC3 siRNA notably decreased the expression of NLRP6, cleavedcaspase-1, cleaved-IL-1β, and GSDMD-N.These results indicated that BRCC3 intervention can effectively reduce inflammation and pyroptosis after cerebral I/R injury.Immunofluorescence staining for caspase-1 and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay revealed that the amount of TUNEL-and caspase-1-positive cells (pyroptosis) remarkably increased in the MCAO group compared to that in the Sham group (Figure 3G).Compared to in the MCAO+NC group, pyroptosis in the BRCC3 siRNA group significantly reduced.Simultaneously, we employed OGD/R to simulate the cerebral I/R injury model in vitro and detected the relevant inflammatory and pyroptosis indicators (Figure 3H-M), consistent with those in vivo.In conclusion, endogenous BRCC3 influences inflammation and pyroptosis following cerebral I/R injury.
24 h after MCAO and OGD/R, and BRCC3 in mouse brain tissue is mainly distributed in neurons; (2) BRCC3 knockdown improved neurological function 24 h after MCAO; (3) BRCC3 knockdown improved inflammation and pyroptosis both in vivo and in vitro; (4) in vitro, NLRP6 knockdown reversed the impact of BRCC3 overexpression on inflammation and pyroptosis; (5) BRCC3 interacts with NLRP6 in vivo and in vitro, both at the N-and C-terminal.Simultaneously, BRCC3 affected the ubiquitination of NLRP6, and (6) BRCC3 promoted NLRP6 inflammasome assembly.
indicate that endogenous BRCC3 siRNA significantly downregulated the protein expression of NLRP6, cleaved-caspase-1, cleaved-IL-1β, and GSDMD-N and reduced neutrophil infiltration, thus improving neurological deficit after MCAO in mice.Conversely, BRCC3 overexpression increased the protein expression of cleaved-caspase-1, cleaved-IL-1β, and GSDMD-N; NLRP6 siRNA reversed these effects of BRCC3-OE.Our findings indicated that the effect of BRCC3 on neuroinflammation and pyroptosis is associated with NLRP6 inflammasome activation in cerebral I/R injury.The specific regulatory relationship between BRCC3 and NLRP6 inflammasome is yet to be determined.The deubiquitination enzyme Cyld can prevent excessive IL-18 production in the colon mucosa via deubiquitination of NLRP6 and inhibition of NLRP6-ASC binding, which regulates IL-18 maturation. 33Notably, BRCC3 deubiquitinated NLRP6; however, BRCC3 facilitated NLRP6 and ASC assembly, inconsistent with the role of Cyld.Moreover, the F I G U R E 5 BRCC3 interacts with NLRP6.(A) CO-IP analysis of interaction between BRCC3 and NLRP6 in HEK293T cells by transfecting NLRP6 and BRCC3 plasmids.(B) CO-IP analysis of interaction between full-length or truncated BRCC3 and NLRP6 in HEK293T cells by transfecting NLRP6 and full-length or truncated BRCC3 plasmids.(C) IP analysis of NLRP6 ubiquitination by transfecting BRCC3, NLRP6, and ubiquitin plasmids in HEK293T cells.(D) IP analysis of NLRP6 ubiquitination by transfecting NLRP6, full-length or truncated BRCC3, and ubiquitin plasmids in HEK293T cells.(E) CO-IP analysis of interaction between BRCC3 and NLRP6 in HT22 cells of the Control and OGD/R groups.(F)Immunofluorescence analysis of BRCC3 and NLRP6 localization in HT22 cells of the Control and OGD/R groups.(G) CO-IP analysis of interaction between BRCC3 and NLRP6 in the Sham and MCAO group.(H) Immunofluorescence analysis of BRCC3 and NLRP6 localization in the Sham and MCAO groups.knockdown or overexpression of BRCC3 significantly affected the expression of NLRP6, whereas Cyld showed no effect on the NLRP6 expression.Therefore, the effect of BRCC3 on NLRP6 may be inconsistent with that of Cyld, requiring further study.Previous literature has shown that BRCC3 has deubiquitination function, 34 but only the full-length function of BRCC3 has been studied, but the specific domain of BRCC3 has not been studied.Therefore, according to the structural characteristics of BRCC3, we constructed N-terminal and C-terminal truncated bodies to explore the N-terminal and C-terminal deubiquitination function.

F I G U R E 6
Effect of BRCC3 on the activation of NLRP6 inflammasome.(A) CO-IP analysis of binding between ASC and NLRP6 in HEK293T cells by transfecting NLRP6, BRC3, and ASC plasmids.(B) CO-IP analysis of binding between ASC and NLRP6 in HEK293T cells by transfecting NLRP6, full-length or truncated BRCC3, and ASC plasmids.(C) CO-IP analysis of binding between ASC and NLRP6 in the Sham, MCAO, MCAO+NC, and MCAO+si-BRCC3 groups.